Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Reexamination Certificate
1999-06-18
2001-08-28
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
C436S526000, C436S534000, C436S536000, C436S825000, C530S391300
Reexamination Certificate
active
06281021
ABSTRACT:
The invention concerns methods for the detection of an analyte in a sample by chemiluminescence or electrochemiluminescence using derivatized test reagents. In addition new reagents and reagent kits for chemiluminescence and electrochemiluminescence detection methods are disclosed.
The detection of analytes by chemiluminescence and electrochemiluminescence is known. Thus EP-A-0 178 450 discloses conjugates of ruthenium complexes and immunologically active materials which are used as reagents in chemiluminescence detection methods. The ruthenium complexes contain three identical or different bicyclic or polycyclic ligands with at least two heterocycles containing nitrogen and at least one of these ligands is substituted by a group such as —SO
3
H or —COOH which renders it water-soluble and at least one of these ligands is directly substituted or substituted via a spacer group with at least one reactive group such as —COOH and the ligands are bound to the ruthenium via nitrogen atoms.
Furthermore the use of metal complexes as labelling reagents for an electrochemiluminescence detection method is known (cf. e.g. EP-A-0 580 979, WO 87/06706, U.S. Pat. No. 5,238,108 or U.S. Pat. No. 5,310,687). Such a chemiluminescence or electrochemiluminescence detection method is based on the conversion of the central atom of the metal complex e.g. ruthenium into the excited MLCT triplet state by chemical or electrochemical means in a suitable measuring device by means of an energy consuming process e.g. electron transfer from a cosubstrate such as a tripropylamine radical. From this excited state it can relax via a forbidden triplet-singlet transition into the ground state with emission of a photon (cf. e.g. WO 90/05296; Leland and Powell, J. Electrochem. Soc. 137 (1990), 3127-3131; Blackburn et al., Clin. Chem. 37 (1991), 1534-1539). However, this reaction mechanism is susceptible to numerous interferences depending on the conditions before and during the measuring phase. Thus secondary reactions can reduce the light yield even to the extent of complete quenching.
It was surprisingly discovered within the framework of the present invention that readily oxidizable chemical groups in components of the test reagent such as free amino groups can interfere with the luminescence reaction. Experiments carried out by the inventors showed that the interfering groups can be present on the molecule carrying the chemiluminescent labelling group or on separate molecules.
The inventors recognized that these oxidizable groups which interfere with the chemiluminescence detection reaction can be blocked (capped) by chemical derivatization and made inaccessible for a chemical or electrochemical reaction without concurrent unacceptable losses of sensitivity in a detection method. In particular the experimental finding is surprising and new that derivatized compounds and in particular those which carry a large number of chemiluminescent labelling groups emit more light than corresponding underivatized compounds and that in an electrochemiluminescence detection method the catalytic anodic current component of derivatized compounds which leads to an undesired reduction in the measured signal is smaller in the case of derivatized compounds than with underivatized compounds. Hence significant advantages can be achieved by using derivatized test reagents in chemiluminescence and electrochemiluminescence detection methods.
Hence a subject matter of the invention is a method for the detection of an analyte in a sample by chemiluminescence or electrochemiluminescence in which the sample is contacted with a test reagent which contains a chemiluminescent or electrochemiluminescent marker group coupled to an analyte-specific receptor and other test components and in which the method is characterized in that oxidizable chemical groups on the labelled receptor or/and on other test components are at least partially protected by derivatization.
The method according to the invention can be a chemiluminescence method i.e. a signal originating from a labelling group is generated by chemical agents. However , the method is preferably an electrochemi-luminescence method in which the light signal originating from the marker group is generated by electrochemical means. An electrochemiluminescence detection method can be carried out in known measuring devices such as a measuring chamber for holding a measuring electrode, means for feeding in and discharging liquids from the measuring chamber and means for detecting the electrochemi-luminescence generated from the measuring chamber. In addition the device can preferably have magnetic means for immobilizing magnetic particles in the sample liquid on the measuring electrode. Such measuring devices are for example described by N. R. Hoyle: “The Application of Electrochemiluminescence to Immunoassay-based Analyte Measurement”, in Bioluminescence and Chemiluminescence; Proceedings of the
8
th International Symposium on Bioluminescence and Chemiluminescence, Cambridge, Sep. 1994, A.K. Campbell et al., (publisher), John Wiley & Sons as well as in WO 89/10551 or W090/11511.
The method according to the invention comprises the use of test reagents in which oxidizable chemical groups which interfere with the chemiluminescence or electrochemiluminescence detection reaction are at least partially protected by derivatization i.e. no or significantly fewer oxidizable groups are converted under the test conditions. The oxidizable chemical groups are for example selected from primary amino groups, secondary amino groups, Schiff bases, thiol groups, acetal groups and ketal groups. Primary or/and secondary aliphatic or aromatic, in particular aliphatic amino groups are particularly preferred.
The oxidizable groups can be derivatized by any known methods. A particularly preferred method for derivatization is acylation especially in the case of primary or secondary amino groups. Other methods of derivatization comprise for example imide formation e.g. the introduction of a phthalimide group (starting from phthalic acid anhydride or O-methoxycarbonylbenzoyl chloride or N-ethoxycarbonylphthalimide), carbamate formation (carbamoylation) and in particular sulfonylation (e.g. the introduction of benzene-sulfonamide or p-toluenesulfonamide groups). In addition the formation of quarternary ammonium salts is also possible.
Known acylation reagents such as carboxylic acid chlorides, anhydrides or active esters can be used for the acylation. Anhydrides or/and active esters are preferred. Active esters such as N-hydroxysuccinimide esters and above all soluble derivatives thereof e.g. sulfonic acid residues are particularly preferred. Furthermore acetic acid p-nitrobenzyl esters also come into consideration. The acyl groups used for the derivatization can be preferably short-chain acyl groups such as acetyl or/and propionyl residues. On the other hand the use of hydrophilic groups e.g. acyl residues carrying hydroxyl, ether or carboxylic acid groups such as tartryl or succinyl residues or polyethyleneglycol-modified acyl residues, is also preferred.
The derivatized substances according to the invention are usually biological substances such as those that are used as test components in detection methods to determine analytes. The derivatized substances can for example be selected from cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, nucleic acid analogues, oligosaccharides, polysaccharides, lipopoly-saccharides, cellular metabolites, haptens, hormones, pharmacological active substances, alkaloids, steroids, vitamins and amino acids. The derivatized substances are preferably selected from peptides, polypeptides, nucleic acids, nucleic acid analogues such as peptidic nucleic acids (PNA), sugars and haptens i.e. organic molecules with a molecular weight of 150 to 2000. The derivatized substances are particularly preferably selected from polypeptides such as antibodies or antibody fragments.
Metal complexes having a structure of the general formula (I) ar
Egger Martin
Josel Hans-Peter
Punzmann Gabriele
Amick Marilyn L.
Ceperley Mary E.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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